Preventing retinal homocysteine dysregulation protects against retinal ganglion cell death

Abstract

Glaucoma, the most common cause of irreversible blindness, is characterized by progressive dysfunction and loss of retinal ganglion cells. There are no neuroprotective therapies. Metabolic dysfunction is an emerging pathophysiological mechanism in glaucoma with widespread changes to energy homeostasis, yet little is known about non‐energy metabolism. We recently identified elevated retinal homocysteine as a strong early metabolomic signature in a rat model of glaucoma. We tested whether local elevation of homocysteine to the retina compounded glaucoma in the rat model by artificially rising homocysteine through intravitreal injection. Short‐term elevation of homocysteine (from 1 to 15 μM, as has been detected in the vitreous of diabetic retinopathy patients) either alone or in addition to glaucoma does not worsen retinal ganglion cell death, suggesting that local elevation of homocysteine does not contribute to retinal ganglion cell degeneration, but may be an indicator of wider metabolic dysfunction. Homocysteine is central to one‐carbon metabolism, an anabolic pathway with an essential role in generation of methionine and methyl donors (necessary for the epigenetic regulation of gene expression), and glutathione (antioxidant). In a mouse model of glaucoma, we identified early‐ and sustained‐dysregulation of genes involved in one‐carbon metabolism in whole retina. In retinal ganglion cells, a number of these genes change prior to detectable neurodegeneration. These pathways require vitamin B6 and B12 as essential cofactors, and folic acid (B9) and choline are essential precursors, for the metabolism of homocysteine. Genes involved in the transport and utilization of these cofactors and precursors were also significantly dysregulated in the retina, further supporting dysfunctional homocysteine metabolism. Dietary deficiency in these leads to homocysteine accumulation and are well associated with optic neuropathies. We hypothesize that local retinal deficiency in these may contribute to dysfunction in one‐carbon metabolism and impact wider retinal ganglion cell homeostasis. To test this hypothesis, we first supplemented the drinking water of mice with B6 (4.5 mg/kg), folic acid (B9) (1.5 mg/kg), cobalamin (B12) (20 μg/kg), and choline (750 mg/kg) as a prophylactic treatment 7 days prior to intravitreal homocysteine injection. We used a supraphysiological dose of homocysteine (500 μM) that causes spontaneous death of ~10% of retinal ganglion cell death after 7 days in untreated animals. Pre‐treated animals were completely protected from retinal ganglion cell death. Next, we pre‐treated rats in the same way for 7 days prior to induction of our glaucoma model. In untreated rats there was a ~ 40% loss of retinal ganglion cells after 14 days, and this was significantly mitigated in pre‐treated rats (only ~25% loss), demonstrating that these cofactors can provide moderate neuroprotection in a complex, multifactorial neurodegenerative context.